Production of C- and N-terminus-specific anti-(GAP-43) antibodies.

the molecule(s) recognized by all four of the antibodies are consumed in the clotting process and that this molecule(s) is fibrin(ogen). AHF2: 1 reacted with fibrinogen separated in SDS/polyacrylamide-gel electrophoresis under reducing conditions followed by Western blotting on to nitrocellulose to reveal two bands of molecular mass 60.5 kDa and 64.4 kDa. These bands correspond to the A-a bands of fibrinogen which can have different molecular masses due to varying degrees of proteolytic degradation [ 11. Western blotting of pooled Dand E-fragments showed reaction for AHF2:l with the E-fragment and a band of molecular mass 61.7 kDa. The nature of the latter band is unclear. Since AHF2:l recognizes both the A-a chain and the E-fragment of fibrinogen, the tentative location of the epitope involved can be narrowed down to amino acid residues 1-78 of the A-a chain which are common to both the A-a chain and the E-fragment. AHF2:2 failed to bind to any chains of fibrinogen when examined by Western blot analysis of reduced fibrinogen in SDS/polyacrylamide-gel electrophoresis, indicating that the epitope( s) recognized by this antibody is structural and present only in the native protein. Probing of fibrinogen and its early plasmin degradation products under non-reducing conditions revealed bands corresponding to fibrinogen and the X-, Yand D-fragments. It did not detect the E-fragment. AHF2:3 antibody reacted with the B-/3 chain of fibrinogen on examination by Western blotting under reducing conditions. It also reacted with the D and E plasmin degradation fragments of fibrinogen. The D-fragment appeared as multiple bands which is in keeping with the findings of Haverkate & Timan [ 21. These heterogeneous bands are probably the result of varying degrees of degradation o f the fibrinogen molecule. Since AHF2:3 binds to both the E-fragment and to the B-B chains. thc tentative location o f the epitope involved can be narrowed to amino acid residues 54-122 of the B-P chain. This is the only portion o f the B-/3 chain shared in common with the E-fragment. A similar epitope must also exist in the D-fragment. Western blot analysis using AHF2:4 showed that this antibody reacted with the B-/3 chain of fibrinogen under reducing conditions and was also seen to react with the D-fragment under non-reducing conditions. The tentative location of the epitope involved can therefore be narrowed to amino acid residues 134-46 1 o f the B-@ chain of fibrinogen. The four monoclonal antibodies described here may find a role in the assay of plasma levels of fibrinogen and its degradation products. It has been noted that elevated levels of fibrin degradation products in patients with unstable angina may be indicative of the onset of acute myocardial infarction [S]. The administration of anti-fibrinolytic agents at the time of observing such an increase in fibrin degradation products may help to prevent the onset of the infarction. A second potential application is in the localization of fibrin deposits (clots) in vivo and the targetting o f fibrinolytic therapy.